1. Field of the Invention
The present invention relates to diagnostic and prognostic markers of endometrial irregularities, particularly infertility. More specifically, the present invention relates to diagnostic markers which can be used in treatment of infertility, kits for timing conception, contraception, and for treatment of excessive bleeding conditions.
2. Background Art
Human endometrium is a unique tissue that undergoes sequential phases of proliferation, secretory changes, tissue shedding and bleeding during menstruation. After ovulation, during a defined period designated as “period of endometrial receptivity” or “implantation window”, a number of sequential changes at the structural and molecular levels make human endometrium susceptible to implantation (Tabibzadeh et al., 1995).
In humans, the ovum is fertilized in the Fallopian tube. The fertilized ovum starts to divide, migrates through the Fallopian tube and enters the uterine cavity around the 3rd to 4th day after ovulation. The blastocyst remains free floating within the endometrial cavity for a day and initiates implantation on day 5-10 after ovulation (Hertig et al., 1956; Formigli et al., 1987; Rogers et al., 1989; Navot et al., 1989; Navot et al., 1991). The identity of the members of the molecular repertoire that make the endometrium receptive to the implantation process still remains largely unknown. If implantation does not occur, however, a second series of changes lead to menstrual shedding of human endometrium. A member of this premenstrual molecular repertoire was recently identified (Kothapalli et al., 1997; Tabibzadeh et al., 1997). The expression of this novel gene was confined to the endometrium immediately prior to and during menstrual bleeding and hence it was originally designated as endometrial bleeding associated factor (ebaf) (Kothapalli et al., 1997). In fact, consistent with its intimate relation with endometrial bleeding, the expression of the gene was found in the endometrium during abnormal uterine bleeding (Kothapalli et al., 1997).
The deduced amino acid sequence of ebaf showed a great amount of identity and similarity with the known members of the TGF-β superfamily. A motif search revealed that the predicted ebaf protein contains most of the conserved cysteine residues of the TGF-β related proteins (Kothapalli et al., 1997) which are necessary for the formation of the cysteine knot structure (Kingsley, D. M., 1994; Daopin et al., 1992). The ebaf sequence contains an additional cysteine residue, 12 amino acids upstream from the first conserved cysteine residue. The only other TGF-β superfamily members, known to contain an additional cysteine residue, are TGF-βs, inhibins and GDF-3 (Kingsley, D. M., 1994; McPherron et al., 1993). ebaf, similar to lefty, GDF-3/Vgr2 and GDF-9, lacks the cysteine residue necessary for the formation of intermolecular disulfide bond (McDonald et al., 1993; McPherron et al., 1993; Jones et al., 1992). Additionally, whereas the carboxy terminus of the TGF-β family is usually CX1CX1, ebaf has a longer C terminal sequence, CX1CX19 (Meno et al., 1996). Therefore, ebaf appears to be an additional member of the TGF-β super family with an unpaired cysteine residue which may not exist as a dimer (Tabibzadeh et al., 1997).
A gene which is called lefty/stra3 of the TGF-β super family is expressed during development in the left side of the mouse embryo in the mesenchyme (Meno et al, 1996; Boullet et al., 1995). The deduced amino acid sequence of ebaf protein is 77% identical and 83% similar to lefty (Meno et al, 1996). Therefore, lefty may be the mouse homolog of the human ebaf or a closely related molecule (Kothapalli et al., 1997; Meno et al, 1996).
Implantation is a complex process which requires interaction of the blastocyst and subsequently the developing embryo and placenta with the endometrium. Initially during this process, blastocyst establishes contact with the surface epithelium of endometrium. Subsequently, during a series of exquisitely controlled steps, the blastocyst is gradually implanted in the underlying stroma. Formation of placenta, the so-called placentation completes the implantation process and establishes a means of supporting the embryo to the end of the pregnancy period.
Most of the information regarding the phases of human implantation are derived from the specimens available in the Carnegie collections. Based on this material, implantation has been divided into various stages (Table 1). At the stage 4a, trophoblasts in different species, use one of the following modes of invasion of endometrium (Schlafke and Enders, 1975):    1. Displacement penetration (mouse, rat): In this mode of endometrial invasion, surface epithelial cells detach from their basement membrane and from each other, they degenerate and then are phagocytozed by trophoblasts. As a consequence, process of implantation is initiated by the exposure of the trophoblasts to the bare underlying basement membrane.    2. Fusion penetration (rabbit, ruminants): In this type of implantation, sycytiotrophoblasts fuse with the surface epithelial cells and form a syncytium that penetrates the basement membrane of the surface epithelium.    3. Intrusion penetration (carnivores): In this type of implantation, the processes of the syncytiotrophoblasts penetrate between the surface epithelial cells and junctions are formed between the trophoblasts and the epithelial cells. The trophoblasts interposed among epithelial cells gradually penetrate through the underlying basement membrane of the surface epithelium.